Project Summary/Abstract Little is known about the airway brush cell compared to its airway epithelium neighbors: basal stem cells, secretory cells, ciliated cells, and neuroendocrine cells. Fundamentally, we do not know its origin, how it is maintained in the adult, or its role in the epithelium, primarily because no genetic tools have been identified to directly characterize this unique, rare epithelial cell type. Using single-cell sequencing and antibody screens, we identify DCLK1 as a novel marker of airway brush cells, enabling us to leverage existing genetic tools to interrogate this cell type's origin, maintenance, and function. Moreover, we identified signature genes in the brush cell that suggest important sensory and immune functions. Here we will address the embryonic origin of the brush cell using an inducible SOX2-CreER endoderm driver and tdTomato reporter. If brush cell are labeled in this mouse by tdTomato, then they are endoderm in origin. In parallel, I will use WNT1-Cre neural crest driver and tdTomato reporter to label neural crest cells during embryonic development. If brush cells are labeled in this mouse by tdTomato, then brush cells originate from neural crest. I will test how the brush cell is maintained in the adult mouse using the newly identified brush cell specific driver, DCLK1-CreER and tdTomato reporter, to label brush cells and their progeny. I will also label basal stem cells and their progeny using basal stem cell specific KRT5-CreER and tdTomato reporter and test to see if they make brush cells. Both of these experiments will be done with chronic exposure of BrdU to ensure that every cell that has proliferated is marked. Antibody detection of brush cells with tdTomato in these lineage strains will allow us to distinguish between stem cell differentiation and self-duplication models of brush cell maintenance. The BrdU experiment will allow us to conclude if brush cells have undergone replication at any time during the lineage tracing. In the case of no BrdU incorporation into brush cells, these results would indicate that, once generated in the embryo brush cells persist, but are not actively renewed. My final aim will define the role of brush cells in the uninjured epithelium by removing the brush cell altogether from the airway epithelium by genetic ablation. Analysis of tissue depleted of brush cells will be phenotypically assessed to determine how the tissue is affected in their absence. I will use the same platform to investigate the role of brush cells in a model of allergic asthma. In completing these aims, I hope to open new avenues for research in cellular mechanisms for environmental sensing, airway epithelial biology, and airway disease.